Fig. 2

Strategies of EV isolation and miRNA characterization in microglia. The left panel shows the strategy to isolate EVs from microglia-conditioned medium with ultracentrifugation procedure (UC procedure). After isolation of microglia from leech CNS, the cells are placed in primary culture. Microglia, cells debris and apoptotic bodies were removed from medium by successive centrifugation steps. Supernatant from the last centrifugation step was ultracentrifuged to pellet EVs and molecular aggregates. The right panel shows the different approaches to identify and validate the presence of miRNAs in microglia EVs. From EVs isolated with UC procedure, a RNAseq analysis allowed the identification of 38 RNA candidates. An additional step in the isolation procedure consisting in an Optiprep™ density gradient (ODG) was added. From new microglia EV isolates, tailing control and RT-PCR using RNA-specific primers against 38 RNA candidates selected 21 putative mature miRNAs. The RNAse A digestion of EV-positive ODG fractions identified 6 miRNAs in microglial EVs. A final method using UC coupled to Size Exclusion Chromatography (SEC) and RNase A treatment confirmed the characterization of the 6 miRNAs in the microglial EVs